Malaria vaccine

ABSTRACT

The present invention provides a particle comprising a polypeptide and at least one malaria antigen, and a composition or vaccine comprising thereof, its use in medicine, particularly in the prevention or treatment of malaria infections.

This application claims benefit from U.S. Provisional Application No.61/830,436 filed Jun. 3, 2013 and U.S. Provisional Application No.61/906,583 filed Nov. 20, 2013.

TECHNICAL FIELD

The present invention relates to a particle comprising a polypeptide andat least one malaria antigen, and a composition or vaccine comprisingthereof, its use in medicine, particularly in the prevention ortreatment of malaria.

BACKGROUND

Malaria is one of the world's most prevalent serious infectiousdiseases, with approximately 250 million cases and 1 million deaths peryear (WHO, 2009). Mortality is primarily in children under the age offive and in pregnant women. Every 45 seconds, an African child dies ofmalaria. The disease is transmitted from person to person by infectedmosquitoes, so past eradication efforts involved massive insecticidecampaigns. These were successful in the Southeast U.S. for example, butfailed in most poorly developed tropical countries. Current effortsinvolve distribution of bednets, particularly bednets impregnated withinsecticide, to prevent mosquito bites at night. However, resistance toinsecticides and to anti-malarial drugs for both prevention andtreatment is rapidly rising. Thus, the need for a malaria vaccine isimperative for protection of millions of people from disease(http://www.globalvaccines.org/content/malaria+vaccine+program/19614).

Malaria caused by Plasmodium falciparum remains a major public healththreat, especially among children and pregnant women in Africa. Aneffective malaria vaccine would be a valuable tool to reduce the diseaseburden and could contribute to elimination of malaria in some regions ofthe world. Current malaria vaccine candidates are directed against humanand mosquito stages of the parasite life cycle, but thus far, relativelyfew proteins have been studied for potential vaccine development.

The most advanced vaccine candidate, RTS,S, conferred partial protectionagainst malaria in phase II clinical trials and is currently beingevaluated in a phase III trial in Africa. (The Journal of ClinicalInvestigation 120(12) 4168-4178, 2010).

The CSP is the predominant surface antigen on sporozoites. CSP iscomposed of an N-terminal region that binds heparin sulfateproteoglycans (RI), a central region containing a four-amino-acid (NPNA)repeat, and a GPI-anchored C-terminal region containing athrombospondin-like domain (RII). The region of the CSP included in theRTS,S vaccine includes the last 16 NPNA repeats and the entire flankingC-terminus. HBsAg particles serve as the matrix carrier for RTS,S, 25%of which is fused to the CSP segment (The Journal of ClinicalInvestigation 120(12) 4168-4178, 2010).

In a series of phase II clinical trials for RTS,S, 30%-50% ofmalaria-naive adults immunized with RTS,S were protected againstchallenge by mosquitoes infected with the homologous P. falciparumclone. In phase II field trials in the Gambia and Kenya, RTS,S conferredshort-lived protection against malaria infection in approximately 35% ofadults, although results from the Kenya trial did not reach statisticalsignificance. Approximately 30%-50% of children and infants immunizedwith RTS,S in phase II trials conducted in Mozambique, Tanzania, andKenya were protected from clinical malaria, however, protection wasgenerally short-lived (The Journal of Clinical Investigation 120(12)4168-4178, 2010). Results from a pivotal, large-scale Phase III trial,published Nov. 9, 2012, online in the New England Journal of Medicine(NEJM), show that the RTS,S malaria vaccine candidate can help protectAfrican infants against malaria. When compared to immunization with acontrol vaccine, infants (aged 6-12 weeks at first vaccination)vaccinated with RTS,S had one-third fewer episodes of both clinical andsevere malaria and had similar reactions to the injection.

There are currently no licensed vaccines against malaria. Highlyeffective malaria vaccine is strongly desired.

Virus-like particles (VLPs) are multiprotein structures that mimic theorganization and conformation of authentic native viruses but lack theviral genome, potentially yielding safer and cheaper vaccine candidates.A handful of prophylactic VLP-based vaccines is currently commercializedworldwide: GlaxoSmithKline's Engerix® (hepatitis B virus) and Cervarix®(human papillomavirus), and Merck and Co., Inc.'s Recombivax HB®(hepatitis B virus) and Gardasile (human papillomavirus) are someexamples. Other VLP-based vaccine candidates are in clinical trials orundergoing preclinical evaluation, such as, influenza virus, parvovirus,Norwalk and various chimeric VLPs. Many others are still restricted tosmall-scale fundamental research, despite their success in preclinicaltests. The implications of large-scale VLP production are discussed inthe context of process control, monitorization and optimization. Themain up- and down-stream technical challenges are identified anddiscussed accordingly. Successful VLP-based vaccine blockbusters arebriefly presented concomitantly with the latest results from clinicaltrials and the recent developments in chimeric VLP-based technology foreither therapeutic or prophylactic vaccination (Expert Rev. Vaccines9(10), 1149-1176, 2010).

Chikungunya virus (CHIKV) has infected millions of people in Africa,Europe and Asia since this alphavirus reemerged from Kenya in 2004. Theseverity of the disease and the spread of this epidemic virus present aserious public health threat in the absence of vaccines or antiviraltherapies. It is reported that a VLP vaccine for epidemic Chikungunyavirus protects non-human primates against infection (Nat Med. 2010March; 16(3): 334-338). US patent publication No. 2012/0003266 disclosesa virus-like particle (VLP) comprising one or more Chikungunya virusstructural polypeptides which is useful for formulating a vaccine orantigenic composition for Chikungunya that induces immunity to aninfection or at least one symptom thereof. WO2012/106356 disclosesmodified alphavirus or flavivirus virus-like particles (VLPs) andmethods for enhancing production of modified VLPs for use in theprevention or treatment of alphavirus and flavivirus-mediated diseases.(these cited references are herein incorporated by reference).

SUMMARY OF THE INVENTION

In the first aspect, the present invention provides a particle which iscapable of being self-assembled, comprising a polypeptide and at leastone malaria antigen, wherein said polypeptide comprises at least onefirst attachment site and said at least one malaria antigen comprises atleast one second attachment site, and wherein said polypeptide and saidmalaria antigen are linked through said at least one first and said atleast one second attachment site.

In the second aspect, the present invention provides a nucleic acidmolecule which is designed for expression of a particle provided in thefirst aspect of the present invention.

In the third aspect, the present invention provides a composition orvaccine comprising the particle provided in the first aspect of thepresent invention and/or the nucleic acid molecule provided in thesecond aspect of the present invention.

In the fourth aspect, the present invention provides a method ofproducing an antibody, comprising contacting the particle provided inthe first aspect of the present invention and/or the nucleic acidmolecule provided in the second aspect of the present invention to amammal.

In the fifth aspect, the present invention provides a method ofimmunomodulation, a method of treating malaria, a method of inducingand/or enhancing immune response against a malaria antigen in a mammal,comprising administering the composition provided in the third aspect ofthe present invention to a mammal.

In sixth aspect, the present invention provides a method of passiveimmunization against a malaria-causing pathogen, comprisingadministering the antibody provided in the fourth aspect of the presentinvention to a mammal.

In seventh aspect, the present invention provides a method of presentingan antigen on macrophage, comprising contacting the particle provided inthe first aspect of the present invention and/or the nucleic acidmolecule provided in the second aspect of the present invention to amammal.

In eighth aspect, the present invention provides a method for producingthe particle provided in the first aspect of the present invention,comprising preparing a vector which is designed for expression of saidparticle; culturing a cell which is transfected with said vector toexpress said particle; and recovering said particle.

BRIEF DESCRIPTION OF THE INVENTION

FIG. 1 shows pVLP74_15 (VLP_CHI 532 NPNAx6) vector.

FIG. 2 shows pVLP78_15 (VLP_CHI 532 NPNAx25) vector.

FIG. 3 shows pVLP74_25 (VLP_VEEV 519 NPNAx6) vector.

FIG. 4 shows that the serum from individual monkeys immunized withMalaria VLPs after 2 weeks induced high titer of antibodies against CSP.

FIG. 5 shows mean value and SD of the data shown in FIG. 4.

FIG. 6 shows effects of combined immunization of CHIKV VLP and VEEV VLPon induction of antibodies against CSP. In the figure, Adj indicatesadjuvant.

FIG. 7 shows effects of administered VLP fused with no malaria antigenon induction of antibodies against CSP. In the figure, 4 w, 6 w, 10 wand 14 w indicate 4 weeks after immunization, 6 weeks afterimmunization, 10 weeks after immunization and 14 weeks afterimmunization, respectively.

FIG. 8 shows effects of administered VLP fused with malaria antigen oninduction of antibodies against CSP. In the figure, 4 w, 6 w, 10 w and14 w indicate 4 weeks after immunization, 6 weeks after immunization, 10weeks after immunization and 14 weeks after immunization, respectively.

FIG. 9 shows effects of administered VLP fused with malaria antigentogether with adjuvant on induction of antibodies against CSP. In thefigure, 4 w, 6 w, 10 w and 14 w indicate 4 weeks after immunization, 6weeks after immunization, 10 weeks after immunization and 14 weeks afterimmunization, respectively.

FIG. 10 shows schedule of the experiment.

FIG. 11 shows detection of 18S malaria DNA by means of PCR.

DETAILED DESCRIPTION OF THE INVENTION (1) a Particle Comprising aPolypeptide and at Least One Malaria Antigen

In the first aspect, the present invention provides a particle which iscapable of being self-assembled, comprising a polypeptide and at leastone malaria antigen, wherein said polypeptide comprises at least onefirst attachment site and said at least one antigen comprises at leastone second attachment site, and wherein said polypeptide and saidmalaria antigen are linked through said at least one first and said atleast one second attachment site.

As used herein, “a particle which is capable of being self-assembled”refers to a particle formed by at least one constituent which isspontaneously assembled. The constituent may be a polypeptide ornon-peptide chemical compound. In one embodiment, “a particle which iscapable of being self-assembled” may be a particle comprising orconsisting of at least one polypeptide. The at least one polypeptideconsists of one or more kinds of peptide. In one embodiment, saidparticle has a diameter of at least 10 nm, for example, at least 20 nm,preferably at least 50 nm. In one embodiment, molecular weight of saidparticle is from 100 kDa to 100,000 kDa, preferably from 400 kDa to30,000 kDa.

A polypeptide used for the present invention may be spontaneouslyassembled. The polypeptide may be a virus structural polypeptide. Thus,the particle provided by the present invention may be a virus likeparticle.

A virus structural polypeptide may be a naturally occurring viralpolypeptide or modified polypeptide thereof. In one embodiment, themodified polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95% or 98%amino acid sequence identity to a naturally occurring viral structuralpolypeptide including capsid and envelope protein. In one embodiment,the modified polypeptide is a mutant where at most 10% of the aminoacids are deleted, substituted, and/or added to a naturally occurringviral structural polypeptide including capsid and envelope protein.

In one embodiment, virus structural polypeptide used for the presentinvention consists of or comprises capsid and/or envelope protein orfragment thereof. For example, virus structural polypeptide used for thepresent invention consists of or comprises capsid and E2 and E1. Anantigen may be inserted into E2. In one embodiment, a particle providedby the first aspect of the present invention can be formed by assembling240 capsids, 240 E1 proteins and 240 E2 proteins where a malaria antigenis inserted into each of E2 proteins.

Virus structural polypeptide used for the present invention may bederived from Alphavirus or Flavivirus. Thus, the particle provided bythe present invention may be a virus like particle derived fromAlphavirus or Flavivirus. Examples of Alphavirus and Flavivirus include,but not limited to, Aura virus, Babanki virus, Barmah Forest virus(BFV), Bebaru virus, Cabassou virus, Chikungunya virus (CHIKV), Easternequine encephalitis virus (EEEV), Eilat virus, Everglades virus, FortMorgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayarovirus, Me Tri virus, Middelburg virus, Mosso das Pedras virus, Mucambovirus, Ndumu virus, O'nyong-nyong virus, Pixuna virus, Rio Negro virus,Ross River virus (RRV), Salmon pancreas disease virus, Semliki Forestvirus, Sindbis virus, Southern elephant seal virus, Tonate virus,Trocara virus, Una virus, Venezuelan equine encephalitis virus (VEEV),Western equine encephalitis virus (WEEV), Whataroa virus, West Nilevirus, dengue virus, tick-borne encephalitis virus and yellow fevervirus.

Malaria is a disease which human or other animal (e.g. mouse) suffersfrom. Example of malaria include, but are not limited to, a diseasecaused by Plasmodium (P.) species including P. falciparum, P. malariae,P. ovale, P. vivax, P. knowlesi, P. berghei, P. chabaudi and P. yoelii

As used herein, the term “malaria antigen” refers to any antigen orfragment thereof. The term antigen or fragment thereof, means anypeptide-based sequence that can be recognized by the immune systemand/or that stimulates a cell-mediated immune response and/or stimulatesthe generation of antibodies.

According to Scand. J. Immunol. 56, 327-343, 2002, considering the wholeparasite life cycle, there are essentially six targets for a malariavaccine: (1) sporozoites; (2) liver stages; (3) merozoites; (4) infectedRBC; (5) parasite toxins; (6) sexual stages.

Table summarizes the main candidate antigens of each stage identified.

Table 1. Main Vaccine Candidates from the Different Phases of PlasmodiumLife Cycle

Targets Candidate Antigens

-   -   Circumsporozoite protein (CSP)    -   Thrombospondin-related adhesive protein (TRAP)        Sporozoite    -   Sporozoite and liver-stage antigen (SALSA)    -   Sporozoite threonine- and asparagine-rich protein (STARP)    -   CSP    -   Liver-stage antigen (LSA)-1 and -3        Liver Stage    -   SALSA    -   STARP    -   Merozoite surface protein (MSP)-1, -2, -3, -4 and -5    -   Erythrocyte-binding antigen (EBA)-175    -   Apical membrane antigen (AMA)-1        Merozoite    -   Rhoptry-associated protein (RAP)-1 and -2    -   Acidic-basic repeat antigen (ABRA)    -   Duffy-binding protein (DBP) (Plasmodium vivax)    -   Ring erythrocyte surface antigen (RESA)    -   Serine-rich protein (SERP)        Blood stage    -   Erythrocyte membrane protein (EMP)-1, -2 and -3    -   Glutamate-rich protein (GLURP)        Toxins    -   Glycosilphosphatidylinositol (GPI)

TABLE 1 1. Main vaccine candidates from the differentphases of Plasmodium life cycle Targets Candidate antigens Sexual stagesPs25, Ps28, Ps48/45 and Ps230 (Scand. J. Immunol. 56, 327-343, 2002)According to the present invention, one or more antigens listedabove can be used as long as it is formed to a particle. Forexample, a circumsporozoite protein and a fragment thereof can beused as an antigen. Examples of circumsporozoite protein include,but are not limited to, Plasmodium falciparum circumsporozoiteprotein consisting of amino acid sequence described below(SEQ ID No.: 56):Mmrklailsyssflfvealfqeyqcygsssntrvinelnydnagtnlynelemnyygkqenwyslkknsrslgenddgnnnngdngregkdedkrdgnnedneklrkpkhkklkqpgdgnpdpnanpnvdpnanpnvdpnanpnvdpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnvdpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnknnqgngqghnmpndpnrnvdenanannavknnnneepsdkhiegylkkiknsistewspcsvtcgngiqvrikpgsankpkdeldyendiekkickmekcssvfnvvnssiglimvlsflflntr.

In one embodiment, malaria antigen is a Plasmodium falciparumcircumsporozoite protein B cell epitope. Example of Plasmodiumfalciparum circumsporozoite protein B cell epitope may be a repeatsequence of NPNA, including (NPNA)₄₋₃₀ (i.e. 4xNPNA, 5xNPNA, 6xNPNA,7xNPNA, 8xNPNA, 9xNPNA, 10xNPNA, 11xNPNA, 12xNPNA, 13xNPNA 14xNPNA,15xNPNA, 16xNPNA, 17xNPNA, 18xNPNA, 19xNPNA, 20xNPNA, 21xNPNA, 22xNPNA,23xNPNA, 24xNPNA, 25xNPNA, 26xNPNA, 27xNPNA, 28xNPNA, 29xNPNA or30xNPNA).

In one embodiment, malaria antigen is a Plasmodium yoeliicircumsporozoite protein B cell epitope including (QGPGAP)₃₋₁₂.

In one embodiment, malaria antigen is a Plasmodium vivaxcircumsporozoite protein B cell epitope including (ANGAGNQPG)₁₋₁₂.

In one embodiment, malaria antigen is a Plasmodium malariaecircumsporozoite protein B cell epitope including (NAAG)₄₋₃₀.

In one embodiment, malaria antigen is a Plasmodium falciparumcircumsporozoite protein T cell epitope. Example of Plasmodiumfalciparum circumsporozoite protein T cell epitope may beEYLNKIQNSLSTEWSPCSVT (SEQ ID No.:44). (EYLNKIQNSLSTEWSPCSVT)₁₋₆ may bealso used as a malaria antigen.

In one embodiment, malaria antigen is a Plasmodium yoeliicircumsporozoite protein T cell epitope which is YNRNIVNRLLGDALNGPEEK(SEQ ID No.45). (YNRNIVNRLLGDALNGPEEK)₁₋₆ may be also used as a malariaantigen.

The present invention addresses one or more of the above needs byproviding antigens, vectors encoding the antigens, and antibodies (andantibody-like molecules including aptamers and peptides) thatspecifically bind to the antigen, together with the uses thereof (eitheralone or in combination) in the prevention or treatment of malariainfections. As used herein, the term “antibody” refers to moleculeswhich are capable of binding an epitope or antigenic determinant. Theterm is meant to include whole antibodies and antigen-binding fragmentsthereof, including single-chain antibodies. Such antibodies includehuman antigen binding antibody fragments and include, but are notlimited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv),single-chain antibodies, disulfide-linked Fvs (sdFv) and fragmentscomprising either a VL or VH domain. The antibodies can be from anyanimal origin including birds and mammals. Preferably, the antibodiesare mammalian e.g. human, murine, rabbit, goat, guinea pig, camel, horseand the like, or other suitable animals e.g. chicken. As used herein,“human” antibodies include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from animals transgenic for one or morehuman immunoglobulins and that do not express endogenousimmunoglobulins, as described, for example, in U.S. Pat. No. 5,939,598,the disclosure of which is incorporated herein by reference in itsentirety.

The antigen used for the present invention can be modified polypeptidederived from a naturally occurring protein. The modified polypeptide maybe a fragment of the naturally occurring protein. In one embodiment, themodified polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95% or 98%amino acid sequence identity to a polypeptide derived from a naturallyoccurring protein. In one embodiment, the modified polypeptide derivedis a mutant where at most 10% of the amino acids are deleted,substituted, and/or added based on a polypeptide derived from naturallyoccurring protein.

In the particle as provided by the present invention, a polypeptide andan antigen may be linked through at least one first attachment sitewhich is present in the polypeptide and at least one second attachmentsite which is present in the antigen.

As used herein, each of “a first attachment site” and “a secondattachment site” refers to a site where more than one substance islinked each other.

In one embodiment, the polypeptide and the antigen are directly fused.Alternatively, one or two linkers may intervene between N-terminalresidue of the antigen and the polypeptide and/or between C-terminalresidue of the antigen and the polypeptide.

The antigen or the polypeptide can be truncated and replaced by shortlinkers. In some embodiments, the antigen or the polypeptide include oneor more peptide linkers. Typically, a linker consists of from 2 to 25amino acids. Usually, it is from 2 to 15 amino acids in length, althoughin certain circumstances, it can be only one, such as a single glycineresidue.

In one embodiment, a nucleic acid molecule, in which polynucleotideencoding the polypeptide is genetically fused with polynucleotideencoding the antigen, is expressed in a host cell so that the firstattachment site and the second attachment site are linked through apeptide bond. In this case, the polypeptide and the antigen are linkedthrough a peptide bond. Relating to this embodiment, the firstattachment site and/or the second attachment site may be geneticallymodified from the original polypeptide or antigen. For example, thefirst attachment site is modified from the polypeptide so that through alinker peptide including SG, GS, SGG, GGS and SGSG, the polypeptide isconjugated with the antigen.

When the polypeptide are chemically conjugated with the antigen, thefirst attachment site and the second attachment site may be linkedthrough a chemical cross-linker which is a chemical compound.

Examples of the cross-linker include, but are not limited to, SMPH,Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC,SVSB, SIA and other cross-linkers available from the Pierce ChemicalCompany.

In one embodiment, the particle provided by the present inventioncomprises a polypeptide linked to an antigen, wherein spatial distancebetween the N-terminal residue and C-terminal residue of the antigen is30 Å or less when the distance is determined in a crystal of the antigenor a naturally occurring protein containing the antigen or modifiedprotein therefrom.

The antigen used for the present invention can be designed by a personskilled in the art. For example, the antigen used for the presentinvention may be a naturally occurring protein or a fragment thereof.Alternatively, the antigen used for the present invention may be aprotein modified from a naturally occurring protein or a fragmentthereof. A person skilled in the art can design the antigen so thatspatial distance between the N-terminal residue and C-terminal residueof the antigen is 30 Å or less when the distance is determined in acrystal of the antigen or a naturally occurring protein containing theantigen or modified protein therefrom. For example, the antigen used forthe particle provided by the present invention can be designed using afree software including PyMOL (e.g. PyMOL v0.99: http:/www.pymol.org).In one embodiment, the spatial distance between the N-terminal residueand C-terminal residue of the antigen is 30 Å (angstrom) or less, 20 Åor less, or 10 Å or less (e.g. from 5 Å to 15 Å, from 5 Å to 12 Å, from5 Å to 11 Å, from 5 Å to 10 Å, from 5 Å to 8 Å, from 8 Å to 15 Å, from 8Å to 13 Å, from 8 Å to 12 Å, from 8 Å to 11 Å, from 9 Å to 12 Å, from 9Å to 11 Å, from 9 Å to 10 Å or from 10 Å to 11 Å).

Chikungunya Virus Like Particle or a Venezuelan Equine EncephalitisVirus Like Particle

In one embodiment, the present invention provides a Chikungunya viruslike particle or a Venezuelan equine encephalitis virus like particlecomprising a Chikungunya or Venezuelan equine encephalitis virusstructural polypeptide and at least one malaria antigen, wherein saidChikungunya virus structural polypeptide or said Venezuelan equineencephalitis virus structural polypeptide comprises at least one firstattachment site and said at least one malaria antigen comprises at leastone second attachment site, and wherein said Chikungunya or Venezuelanequine encephalitis virus structural polypeptide and said at least oneantigen are linked through said at least one first and said at least onesecond attachment site.

In one embodiment, a spatial distance between the N-terminal residue andC-terminal residue of the malaria antigen may be 30 Å or less; 25 Å orless; 20 Å or less; 15 Å or less; 14 Å or less; 13 Å or less; 12 Å orless; 11 Å or less; 10 Å or less; 9 Å or less; or 8 Å or less (e.g. from5 Å to 15 Å, from 5 Å to 12 Å, from 5 Å to 11 Å, from 5 Å to 11 Å, from5 Å to 8 Å, from 8 Å to 15 Å, from 8 Å to 13 Å, from 8 Å to 12 Å, from 8Å to 11 Å, from 9 Å to 12 Å, from 9 Å to it Å, from 9 Å to 10 Å or from10 Å to 11 Å) when the distance is determined in a crystal of themalaria antigen or a naturally occurring protein containing the malariaantigen or modified protein therefrom.

In one embodiment, the malaria antigen is linked to the Chikungunya orVenezuelan equine encephalitis virus structural polypeptide by way ofchemical cross-linking or as a fusion protein produced by way of geneticengineering.

A Chikungunya or Venezuelan equine encephalitis virus structuralpolypeptide used in the present invention may comprise a Chikungunya orVenezuelan equine encephalitis virus envelope protein and/or a capsid.

Examples of Chikungunya virus include, but are not limited to, strainsof 37997 and LR2006 OPY-1.

Examples of Venezuelan equine encephalitis virus include, but are notlimited to, TC-83.

Chikungunya or Venezuelan equine encephalitis virus structuralpolypeptide used in the present invention may naturally occurring virusstructural polypeptide or modified polypeptide thereof. The modifiedpolypeptide may be a fragment of the naturally occurring virusstructural polypeptide. In one embodiment, the modified polypeptide hasat least 70%, 75%, 80%, 85%, 90%, 95% or 98% amino acid sequenceidentity to a naturally occurring viral capsid and/or envelope protein.In one embodiment, the modified polypeptide is a mutant where at most10% of the amino acids are deleted, substituted, and/or added based on anaturally occurring viral capsid and/or envelope protein. For example,K64A or K64N mutation may be introduced into a capsid of Venezuelanequine encephalitis virus structural polypeptide used in the presentinvention.

Chikungunya or Venezuelan equine encephalitis virus structuralpolypeptide may consist of or comprise a capsid, E2 and E1.

Examples of Chikungunya virus structural polypeptide include, but arenot limited to, Capsid-E2-E1 of Chikungunya virus Strain 37997, andCapsid-E2-E1 of Chikungunya virus LR2006 OPY-1.

Examples of Venezuelan equine encephalitis virus structural polypeptideinclude, but are not limited to, Capsid-E2-E1 of Venezuelan equineencephalitis virus Strain TC-83.

An exemplary Chikungunya virus structural polypeptide sequence isprovided at Genbank Accession No. ABX40006.1, which is described below(SEQ ID No.:1):

mefiptqtfynrryqprpwtprptiqvirprprpqrqagqlaqlisavnkltmravpqqkprrnrknkkqkqkqqapqnntnqkkqppkkkpaqkkkkpgrrermcmkiendcifevkhegkvtgyaclvgdkvmkpahvkgtidnadlaklafkrsskydlecaqipvhmksdaskfthekpegyynwhhgavqysggrftiptgagkpgdsgrpifdnkgrvvaivlgganegartalsvvtwnkdivtkitpegaeewslaipvmcllanttfpcsqppctpccyekepeetlrmlednvmrpgyyqllgasltcsphrqrrstkdnfnvykatrpylahcpdcgeghschspvalerirneatdgtlkiqvslqigiktddshdwtklrymdnhmpadaeraglfvrtsapctitgtmghfilarcpkgetltvgftdsrkishscthpfhhdppvigrekfhsrpqhgkelpcstyvqstaatteeievhmppdtpdrtlmsqqsgnvkitvngqtvrykcncggsnegltttdkvinnckvdqchaavtnhkkwqynsplvprnaelgdrkgkihipfplanvtcrvpkarnptvtygknqvimllypdhptllsyrnmgeepnyqeewvmhkkevvltvpteglevtwgnnepykywpqlstngtahghpheiilyyyelyptmtvvvvsvatfillsmvgmaagmcmcarrrcitpyeltpgatvpfllsliccirtakaatyqeaaiylwneqqplfwlqaliplaalivlcnclrllpcccktlaflavmsvgahtvsayehvtvipntvgvpyktlvnrpgyspmvlemellsvtleptlsldyitceyktvipspyvkccgtaeckdknlpdysckvftgvypfmwggaycfcdaentqlseahveksescktefasayrahtasasaklrvlyqgnnitvtayangdhavtvkdakfivgpmssawtpfdnkivvykgdvynmdyppfgagrpgqfgdiqsrtpeskdvyantqlvlqrpavgtvhvpysqapsgfkywlkergaslqhtapfgcqiatnpvravncavgnmpisidipeaaftrvvdapsltdmscevpacthssdfggvaiikyaaskkgkcavhsmtnavtireaeievegnsqlqisfstalasaefrvqvcstqvhcaaechppkdhivnypashttlgvqdisatamswvqkitggvglvvavaalilivvlcvsfsrh

Another exemplary Chikungunya virus structural polypeptide sequence isprovided at Genbank Accession No. ABX40011.1, which is described below(SEQ ID No.:2):

mefiptqtfynrryqprpwaprptiqvirprprpqrqagqlaqlisavnkltmravpqqkprrnrknkkqrqkkqapqndpkqkkqppqkkpaqkkkkpgrrermcmkiendcifevkhegkvmgyaclvgdkvmkpahvkgtidnadlaklafkrsskydlecaqipvhmksdaskfthekpegyynwhhgavqysggrftiptgagkpgdsgrpifdnkgrvvalvlgganegartalsvvtwnkdivtkitpegaeewslalpvlcllanttfpcsqppctpccyekepestlrmlednvmrpgyyqllkasltcsphrqrrstkdnfvnykatrpylahcpdcgeghschsplalerirneatdgtlkiqvslqiglktddshdwtklrymdshtpadaeragllvrtsapctitgtmghfilarcpkgetltvgftdsrkishtcthpfhheppvigrerfhsrpqhgkelpcstyvqstaataeeievhmppdtpdrtlmtqqagnvkitvnggtvrykcncggsnegltttdkvinnckidqchaavtnhknwqynsplvprnaelgdrkgkihipfplanvtcrvpkarnptvtygknqvtmllypdhptllsyrnmgqepnyheewvthkkevtltvpteglevtwgnnepykywpqmstngtahghpheillyyyelyptmtvvivsvasfvllsmvgtavgmcvcarrrcitpyeltpgatvpfllsllccvrttkaatyyeaaaylwneqqplfwlqaliplaalivlcnvlkllpcccktlaflavmsigahtvsayehvtvipntvgvpyktlvnrpgyspmvlemelqsvtleptlsldyitceyktvipspyvkccgtaeckdkslpdysckvftgvypfmwggaycfcdaentqlseahveksescktefasayrahtasasaklrvlyqgnnitvaayangdhavtvkdakfvvgpmssawtpfdnkivvykgdvynmdyppfgagrpgqfgdiqartpeskdvyantqlvlqrpaagtvhvpysqapsgfkywlkergaslqhtapfgcqiatnpvravncavgnipisidipdaaftrvvdapsvtdmscevpacthssdfggvailkytaskkgkcavhsmtnavtireadvevegnsqlqisfstalasaefrvqvcstqvhcaaachppkdhlvnypashttlgvqdisttamawvqkitggvglivavaalilivvlcvsfsrh.

An exemplary Venezuelan equine encephalitis virus structural polypeptideis provided at Genbank Accession No. L01443.1(http://www.ncbi.nlm.nih.gov/nuccore/L01443.1), which is described below(SEQ ID No.:3):

mfpfqpmypmqpmpyrnpfaaprrpwfprtdpflamqvqeltrsmanltfkqrrdappegpsaakpkkeasqkqkgggqgkkkknqgkkkaktgppnpkaqngnkkktnkkpgkrqrmvmklesdktfpimlegkingyacvvggklfrpmhvegkidndvlaalktkkadkydleyadvpqnmradtfkythekpqgyyswhhgavqyengrftvpkgvgakgdsgrpildnqgrvvaivlggvnegsrtalsvvmwnekgvtvkytpenceqwslvttmcllanvtfpcaqppicydrkpaetlamlsvnvdnpgydelleaavkcpgrkrrsteelfneykltrpymarcircavgschspiaieavksdghdgyvrlqtssqygldssgnlkgrtmrydmhgtikeiplhqvslytsrpchivdghgyfllarcpagdsitmefkkdsvrhscsvpyevkfnpvgrelythppehgveqacqvyahdaqnrgayvemhlpgsevdsslvslsgssvtvtppdgtsalvececggtkisetinktkqfsqctkkeqcrayrlqndkwvynsdklpkaagatlkgklhvpflladgkctvplapepmitfgfrsvslklhpknptylitrqladephythelisepavrnftvtekgwefvwgnhppkrfwaqetapgnphglphevithyyhrypmstilglsicaaiatvsvaastwlfcrsrvacltpyrltpnaripfclavlccartaraettwesldhlwnnnqqmfiiqlliplaalivvtrllrcvccvvpflvmagaagagayehattmpsqagisyntivnragyaplpisitptkikliptvnleyctchyktgmdspaikccgsqectptyrpdeqckvftgvypfmwggaycfcdtentqvskayvmksddcladhaeaykahtasvqaflnitvgehsivttvyvngetpvnfngvkitagplstawtpfdrkivqyageiynydfpeygagqpgafgdiqsrtvsssdlyantnlvlqrpkagaihvpytqapsgfeqwkkdkapslkftapfgceiytnpiraencavgsiplafdipdalftrvsetptlsaaectlnecvyssdfggiatvkysasksgkcavhvpsgtatlkeaavelteqgsatihfstanihpefrlgictsyvtckgdchppkdhivthpqyhaqtftaavsktawtwltsllggsaviiiiglvlativamyvltnqkhn.

In one embodiment, a first attachment site comprises an amino group,preferably an amino group of a lysine residue. In one embodiment, thesecond attachment site comprises sulfhydryl group, preferably, asulfhydryl group of a cysteine.

In one embodiment, a conjugation of more than two substances (e.g.antigen and Chikungunya or Venezuelan equine encephalitis virusstructural polypeptide) through a first attachment site or a secondattachment site is achieved using chemical cross linker. Examples of thecross-linker include, but are not limited to, SMPH, Sulfo-MBS,Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIAand other cross-linkers available from the Pierce Chemical Company.

According to the present invention, a Chikungunya or Venezuelan equineencephalitis virus like particle comprising a Chikungunya or Venezuelanequine encephalitis virus structural polypeptide and an antigen, whereinsaid Chikungunya or Venezuelan equine encephalitis virus structuralpolypeptide and said antigen are expressed as a fusion protein can beprovided.

In one embodiment, the antigen can be fused with any site of theChikungunya or Venezuelan equine encephalitis virus structuralpolypeptide. For example, the antigen may be directly or indirectlylinked to N- or C-terminal of the Chikungunya or Venezuelan equineencephalitis virus structural polypeptide, or the antigen may beinserted into Chikungunya or Venezuelan equine encephalitis virusstructural protein.

In one embodiment, at least one antigen is inserted into E2 ofChikungunya or Venezuelan equine encephalitis virus structural protein.For example, regarding Chikungunya virus structural protein, at leastone antigen is inserted between residues 519 and 520 of SEQ ID Nos.1 or2 (i.e. between G at 519-position and Q at 520-position of SEQ ID Nos.1or 2); between residues 530 and 531 of SEQ ID Nos.1 or 2 (i.e. between Gat 530-position and S at 531-position of SEQ ID Nos.1 or 2); betweenresidues 531 and 532 of SEQ ID Nos.1 or 2 (i.e. between S at531-position and N at 532-position of SEQ ID Nos.1 or 2); betweenresidues 529 and 530 of SEQ ID Nos.1 or 2 (i.e. between G at529-position and G at 530-position of SEQ ID Nos.1 or 2); or betweenresidues 510 and 511 of SEQ ID Nos.1 or 2 (i.e. between S at510-position and G at 511-position of SEQ ID Nos.1 or 2); or betweenresidues 511 and 512 of SEQ ID Nos.1 or 2 (i.e. between G at511-position and N at 512-position of SEQ ID Nos.1 or 2); or betweenresidues 509 and 510 of SEQ ID Nos.1 or 2 (i.e. between Q at509-position and S at 510-position of SEQ ID Nos.1 or 2).

For example, regarding Venezuelan equine encephalitis virus structuralprotein, at least one antigen is inserted between residues 517 and 518of SEQ ID No.3 (i.e. between G at 517-position and S at 518-position ofSEQ ID No.3); between residues 518 and 519 of SEQ ID No.3 (i.e. betweenS at 518-position and S at 519-position of SEQ ID No.3); betweenresidues 519 and 520 of SEQ ID No.3 (i.e. between S at 519-position andV at 520-position of SEQ ID No.3); between residues 515 and 516 of SEQID No.3 (i.e. between L at 515-position and S at 516-position of SEQ IDNo.3); between residues 516 and 517 of SEQ ID No.3 (i.e. between S at516-position and G at 517-position of SEQ ID No.3); between residues 536and 537 of SEQ ID No.3 (i.e. between C at 536-position and G at537-position of SEQ ID No.3); between residues 537 and 538 of SEQ IDNo.3 (i.e. between G at 537-position and G at 538-position of SEQ IDNo.3); between residues 538 and 539 of SEQ ID No.3 (i.e. between G at538-position and T at 539-position of SEQ ID No.3).

The fusion protein may be expressed using a conventional technique inthe art. A variety of expression systems can be used for the expressionof the fusion protein. For example, the fusion protein can be expressedin 293 cells, Sf9 cells or E. coli.

A polypeptide derived from Chikungunya virus (CHIKV) or Venezuelanequine encephalitis virus (VEEV) may be a naturally occurring viralpolypeptide or modified polypeptide thereof. In addition, a polypeptidederived from malaria antigen may be a naturally occurring polypeptide ormodified polypeptide of the naturally occurring polypeptide or afragment of the naturally occurring polypeptide or the modified peptide.The modified polypeptide may be a fragment of the naturally occurringvirus structural polypeptide.

In one embodiment, the modified polypeptide derived from malaria antigenhas at least 70%, 75%, 80%, 85%, 90%, 95% or 98% amino acid sequenceidentity to a naturally occurring polypeptide. In one embodiment, themodified peptide derived from malaria antigen is a mutant where at most10% of the amino acids are deleted, substituted, and/or added based on anaturally occurring polypeptide derived from malaria antigen.

When a polypeptide derived from a virus is conjugated with a polypeptidederived from an antigen, a linker peptide including SG, GS, SGG, GGSSGSG and TRGGS may be used. Examples of conjugation of the polypeptidederived from a virus (referred to as “PFV” below) with the polypeptidederived from the antigen (referred to as “PFA” below) include, but notlimited to: PFV-SG-PFA-GS-PFV; PFV-SG-PFA-GGS-PFV; PFV-SSG-PFA-GS-PFV;PFV-SGG-PFA-GGS-PFV; PFV-SGSG-PFA-GS-PFV; and PFA-SGG-PFA-TRGGS-PFV.

In one embodiment, the present invention provides a virus like particlecomprising a fusion protein of a polypeptide derived from Chikungunyavirus (CHIKV) or Venezuelan equine encephalitis virus (VEEV) and apolypeptide derived from malaria antigen, wherein the virus likeparticle is prepared by transfecting an expression vector comprising anucleic acid molecule corresponding to the amino acid sequencerepresented by SEQ ID NO. 28, 31, 34, 37, 39, 41 or 43 into a mammaliancell (e.g. 293F cell). Regarding this embodiment, modified fusionprotein can be also used for a virus like particle provided by thepresent invention, which can be prepared by transfecting an expressionvector comprising a nucleic acid molecule corresponding to the aminoacid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 98% aminoacid sequence identity to SEQ ID NO. 28, 31, 34, 37, 39, 41 or 43 into amammalian cell (e.g. 293F cell).

In one embodiment, the present invention provides a virus like particlecomprising or consisting of:

one or more capsid of Chikungunya virus (CHIKV) or Venezuelan equineencephalitis virus (VEEV);

one or more E1 of Chikungunya virus (CHIKV) or Venezuelan equineencephalitis virus (VEEV); and

one or more E2 of Chikungunya virus (CHIKV) or Venezuelan equineencephalitis virus (VEEV), wherein malaria antigen is inserted into E2of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus(VEEV). For example, present invention provides a virus like particlecomprising or consisting of:240 capsids of Chikungunya virus (CHIKV) or Venezuelan equineencephalitis virus (VEEV);240 E1s of Chikungunya virus (CHIKV) or Venezuelan equine encephalitisvirus (VEEV); and240 E2s of Chikungunya virus (CHIKV) or Venezuelan equine encephalitisvirus (VEEV), wherein malaria antigen is inserted into each of E2s ofChikungunya virus (CHIKV) or Venezuelan equine encephalitis virus(VEEV).

In this embodiment, the E2 into which the antigen is inserted mayconsist of an amino acid sequence represented by SEQ ID No.50; the E1may consist of an amino acid sequence represented by SEQ ID No.51; andthe capsid may consist of an amino acid sequence represented by SEQ IDNO.: 52; or

the E2 into which the antigen is inserted may consist of an amino acidsequence represented by SEQ ID NO.53; the E1 may consist of an aminoacid sequence represented by SEQ ID NO.54; and the capsid may consist ofan amino acid sequence represented by SEQ ID NO.: 55.

Further, regarding this embodiment, modified capsid of Chikungunya virus(CHIKV) or Venezuelan equine encephalitis virus (VEEV), modified E1 ofChikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV)and modified E2 of Chikungunya virus (CHIKV) or Venezuelan equineencephalitis virus (VEEV) may be used for the virus like particle. Forexample, the modified capsid of Chikungunya virus (CHIKV) or Venezuelanequine encephalitis virus (VEEV) may have at least 70%, 75%, 80%, 85%,90%, 95% or 98% amino acid sequence identity to the amino acid sequencerepresented by SEQ ID NO.: 52 or SEQ ID No.:55; the modified E1 ofChikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV)may have at least 70%, 75%, 80%, 85%, 90%, 95% or 98% amino acidsequence identity to the amino acid sequence represented by SEQ ID NO.:51 or SEQ ID No.:54; and/or the modified E2 of Chikungunya virus (CHIKV)or Venezuelan equine encephalitis virus (VEEV) may have at least 70%,75%, 80%, 85%, 90%, 95% or 98% amino acid sequence identity to the aminoacid sequence represented by SEQ ID NO.: 50 or SEQ ID No.:53.

Also, the modified capsid, E1 or E2 may be a mutant where at most 10% ofthe amino acids are deleted, substituted, and/or added based on thecapsid consisting of an amino acid sequence represented by SEQ ID NO.:52 or SEQ ID No.:55; E1 consisting of an amino acid sequence representedby SEQ ID NO.: 51 or SEQ ID No.:54; and/or E2 consisting of an aminoacid sequence represented by SEQ ID NO.: 50 or SEQ ID No.:53.

(2) Nucleotide, Vector, Host Cell

In the second aspect, the present invention provides a nucleic acidmolecule which is designed for expression of a particle as provided inthe first aspect of the present invention.

In one embodiment, the present invention provides a nucleic acidmolecule comprising a nucleotide sequence that encodes the Chikungunyaor Venezuelan equine encephalitis virus like particle as describedabove.

Examples of the nucleotide sequence that encodes the Chikungunya orVenezuelan equine encephalitis virus like particle include, but are notlimited to, a nucleotide sequence encoding envelope of Chikungunya virusStrain 37997, a nucleotide sequence encoding Capsid-envelope ofChikungunya virus Strain 37997, a nucleotide sequence encoding envelopeof Chikungunya virus Strain LR2006 OPY-1, a nucleotide sequence encodingCapsid-envelope of Chikungunya virus LR2006 OPY-1, a nucleotide sequenceencoding envelope of Venezuelan equine encephalitis virus Strain TC-83and a nucleotide sequence encoding Capsid-envelope of Venezuelan equineencephalitis virus TC-83.

Regarding Chikungunya virus, an exemplary nucleotide sequence thatencodes envelope is described below (SEQ ID No.:4):

Atgagcctcgccctcccggtcttgtgcctgttggcaaacactacattcccctgctctcagccgccttgcacaccctgctgctacgaaaaggaaccggaaagcaccttgcgcatgcttgaggacaacgtgatgagacccggatactaccagctactaaaagcatcgctgacttgctctccccaccgccaaagacgcagtactaaggacaattttaatgtctataaagccacaagaccatatctagctcattgtcctgactgcggagaagggcattcgtgccacagccctatcgcattggagcgcatcagaaatgaagcaacggacggaacgctgaaaatccaggtctctttgcagatcgggataaagacagatgacagccacgattggaccaagctgcgctatatggatagccatacgccagcggacgcggagcgagccggattgcttgtaaggacttcagcaccgtgcacgatcaccgggaccatgggacactttattctcgcccgatgcccgaaaggagagacgctgacagtgggatttacggacagcagaaagatcagccacacatgcacacacccgttccatcatgaaccacctgtgataggtagggagaggttccactctcgaccacaacatggtaaagagttaccttgcagcacgtacgtgcagagcaccgctgccactgctgaggagatagaggtgcatatgcccccagatactcctgaccgcacgctgatgacgcagcagtctggcaacgtgaagatcacagttaatgggcagacggtgcggtacaagtgcaactgcggtggctcaaacgagggactgacaaccacagacaaagtgatcaataactgcaaaattgatcagtgccatgctgcagtcactaatcacaagaattggcaatacaactcccctttagtcccgcgcaacgctgaactcggggaccgtaaaggaaagatccacatcccattcccattggcaaacgtgacttgcagagtgccaaaagcaagaaaccctacagtaacttacggaaaaaaccaagtcaccatgctgctgtatcctgaccatccgacactcttgtcttaccgtaacatgggacaggaaccaaattaccacgaggagtgggtgacacacaagaaggaggttaccttgaccgtgcctactgagggtctggaggtcacttggggcaacaacgaaccatacaagtactggccgcagatgtctacgaacggtactgctcatggtcacccacatgagataatcttgtactattatgagctgtaccccactatgactgtagtcattgtgtcggtggcctcgttcgtgcttctgtcgatggtgggcacagcagtgggaatgtgtgtgtgcgcacggcgcagatgcattacaccatatgaattaacaccaggagccactgttcccttcctgctcagcctgctatgctgcgtcagaacgaccaaggcggccacatattacgaggctgcggcatatctatggaacgaacagcagcccctgttctggttgcaggctcttatcccgctggccgccttgatcgtcctgtgcaactgtctgaaactcttgccatgctgctgtaagaccctggcttttttagccgtattttgagcatcggtgcccacactgtgagcgcgtacgaacacgtaacagtgatcccgaacacggtgggagtaccgtataagactcttgtcaacagaccgggttacagccccatggtgttggagatggagctacaatcagtcaccttggaaccaacactgtcacttgactacatcacgtgcgagtacaaaactgtcatcccctccccgtacgtgaagtgctgtggtacagcagagtgcaaggacaagagcctaccagactacagctgcaaggtctttactggagtctacccatttatgtggggcggcgcctactgcttttgcgacgccgaaaatacgcaattgagcgaggcacatgtagagaaatctgaatcttgcaaaacagagtttgcatcggcctacagagcccacaccgcatcggcgtcggcgaagctccgcgtcctttaccaaggaaacaacattaccgtagctgcctacgctaacggtgaccatgccgtcacagtaaaggacgccaagtttgtcgtgggcccaatgtcctccgcctggacaccttttgacaacaaaatcgtggtgtacaaaggcgacgtctacaacatggactacccaccttttggcgcaggaagaccaggacaatttggtgacattcaaagtcgtacaccggaaagtaaagacgtttatgccaacactcagttggtactacagaggccagcagcaggcacggtacatgtaccatactctcaggcaccatctggcttcaagtattggctgaaggaacgaggagcatcgctacagcacacggcaccgttcggttgccagattgcgacaaacccggtaagagctgtaaattgcgctgtggggaacataccaatttccatcgacataccggatgcggcctttactagggttgtcgatgcaccctctgtaacggacatgtcatgcgaagtaccagcctgcactcactcctccgactttgggggcgtcgccatcatcaaatacacagctagcaagaaaggtaaatgtgcagtacattcgatgaccaacgccgttaccattcgagaagccgacgtagaagtagaggggaactcccagctgcaaatatccttctcaacagccctggcaagcgccgagtttgcgcgtgcaagtgtgctccacacaagtacactgcgcagccgcatgccaccctccaaaggaccacatagtcaattacccagcatcacacaccacccttggggtccaggatatccacaacggcaatgtcttgggtgcagaagattacgggaggagtaggattaattgttgctgttgctgccttaattttaattgtggtgctatgcgtgtcgtttagcaggcac

Regarding Chikungunya virus, another exemplary nucleotide sequence thatencodes envelope is described below (SEQ ID No.:5):

Atgagtcttgccatcccagttatgtgcctgttggcaaacaccacgttcccctgctcccagcccccttgcacgccctgctgctacgaaaaggaaccggaggaaaccctacgcatgcttgaggacaacgtcatgagacctgggtactatcagctgctacaagcatccttaacatgttctccccaccgccagcgacgcagacaccaaggacaacttcaatgtctataaagccacaagaccatacttagctcactgtcccgactgtggagaagggcactcgtgccatagtcccgtagcactagaacgcatcagaaatgaagcgacagacgggacgctgaaaatccaggtctccttgcaaatcggaataaagacggatgacagccacgattggaccaagctgcgttatatggacaaccacatgccagcagacgcagagagggcgggctatttgtaagaacatcagcaccgtgtacgattactggaacaatgggacacttcatcctggcccgatgtccaaaaggggaaactctgacggtgggattcactgacagtaggaagattagtcactcatgtacgcacccatttcaccacgaccctcctgtgataggtcgggaaaaattccattcccgaccgcagcacggtaaagagctaccttgcagcacgtacgtgcagagcaccgccgcaactaccgaggagatagaggtacacatgcccccagacacccctgatcgcacattaatgtcacaacagtccggcaacgtaaagatcacagtcaatggccagacggtgcggtacaagtgtaattgcggtggctcaaatgaaggactaacaactacagacaaagtgattaataactgcaaggttgatcaatgtcatgccgcggtcaccaatcacaaaaagtggcagtataactcccctctggtcccgcgtaatgctgaacttggggaccgaaaaggaaaaattcacatcccgtttccgctggcaaatgtaacatgcagggtgcctaaagcaaggaaccccaccgtgacgtacgggaaaaaccaagtcatcatgctactgtatcctgaccacccaacactcctgtcctaccggaatatgggagaagaaccaaactatcaagaagagtgggtgatgcataagaaggaagtcgtgctaaccgtgccgactgaagggctcgaggtcacgtggggcaacaacgagccgtataagtattggccgcagttatctacaaacggtacagcccatggccacccgcatgagataattctgtattattatgagctgtaccccactatgactgtagtagttgtgtcagtggccacgttcatactcctgtcgatggtgggtatggcagcggggatgtgcatgtgtgcacgacgcagatgcatcacaccgtatgaactgacaccaggagctaccgtccctttcctgcttgcttagcctaatatgctgcatcagaacagctaaagcggccacataccaagaggctgcgatatcctgtggaacgagcagcaacctttgttttggctacaagcccttattccgctggcagccctgattgttctatgcaactgtctgagactcttaccatgctgctgtaaaacgttggcttttttagccgtaatgagcgtcggtgcccacactgtgagcgcgtacgaacacgtaacagtgatcccgaacacggtgggagtaccgtataagactctagtcaatagacctggctacagccccatggtattggagatggaactactgtcagtcactttggagccaacactatcgcttgattacatcacgtgcgagtacaaaaccgtcatcccgtctccgtacgtgaagtgctgcggtacagcagagtgcaaggacaaaaacctacctgactacagctgtaaggtcttcaccggcgtctacccattttgtggggcggcgcctactgcttctgcgacgctgaaaacacgcagttgagcgaagcacacgtggagaagtccgaatcatgcaaaacagaatttgcatcagcatacagggctcataccgcatctgcatcagctaagctccgcgtcctttaccaaggaaataacatcactgtaactgcctatgcaaacggcgaccatgccgtcacagttaaggacgccaaattcattgtggggccaatgtcttcagcctggacacctttcgacaacaaaattgtggtgtacaaaggtgacgtctataacatggactacccgccctttggcgcaggaagaccaggacaatttggcgatatccaaagtcgcacacctgagagtaaagacgtctatgctaatacacaactggtactgcagagaccggctgtgggtacggtacacgtgccatactctcaggcaccatctggctttaagtattggctaaaagaacgcggggcgtcgctgcagcacacagcaccatttggctgccaaatagcaacaaacccggtaagagcggtgaactgcgccgtagggaacatgcccatctccatcgacataccggaagcggccttcactagggtcgtcgacgcgccctctttaacggacatgtcgtgcgaggtaccagcctgcacccattcctcagactttgggggcgtcgccattattaaatatgcagccagcaagaaaggcaagtgtgcggtgcattcgatgactaacgccgtcactattcgggaagctgagatagaagttgaagggaattctcagctgcaaatctctttctcgacggccttagccagcgccgaattccgcgtacaagtctgttctacacaagtacactgtgcagccgagtgccaccccccgaaggaccacatagtcaactacccggcgtcacataccaccctcggggtccaggacatctccgctacggcgatgtcatgggtgcagaagatcacgggaggtgtgggactggttgttgctgttgccgcactgattctaatcgtggtgctatgcgtgtcgttcagcaggcac

Regarding Chikungunya virus, an exemplary nucleotide sequence thatencodes a Capsid-envelope is described below (SEQ ID No.:6):

atggagttcatcccgacgcaaactttctataacagaaggtaccaaccccgaccctgggccccacgccctacaattcaagtaattagacctagaccacgtccacagaggcaggctgggcaactcgcccagctgatctccgcagtcaacaaattgaccatgcgcgcggtacctcaacagaagcctcgcagaaatcggaaaaacaagaagcaaaggcagaagaagcaggcgccgcaaaacgacccaaagcaaaagaagcaaccaccacaaaagaagccggctcaaaagaagaagaaaccaggccgtagggagagaatgtgcatgaaaattgaaaatgattgcatcttcgaagtcaagcatgaaggcaaagtgatgggctacgcatgcctggtgggggataaagtaatgaaaccagcacatgtgaagggaactatcgacaatgccgatctggctaaactggcctttaagcggtcgtctaaatacgatcttgaatgtgcacagataccggtgcacatgaagtctgatgcctcgaagtttacccacgagaaacccgaggggtactataactggcatcacggagcagtgcagtattcaggaggccggttcactatcccgacgggtgcaggcaagccgggagacagcggcagaccgatcttcgacaacaaaggacgggtggtggccatcgtcctaggaggggccaacgaaggtgcccgcacggccctctccgtggtgacgtggaacaaagacatcgtcacaaaaattacccctgagggagccgaagagtggagcctcgccctcccggtcttgtgcctgttggcaaacactacattcccctgctctcagccgccttgcacaccctgctgctacgaaaaggaaccggaaagcaccttgcgcatgcttgaggacaacgtgatgagacccggatactaccagctactaaaagcatcgctgacttgctctccccaccgccaaagacgcagtactaaggacaattttaatgtctataaagccacaagaccatatctagctcattgtcctgactgcggagaagggcattcgtgccacagccctatcgcattggagcgcatcagaaatgaagcaacggacggaacgctgaaaatccaggtctctttgcagatcgggataaagacagatgacagccacgattggaccaagctgcgctatatggatagccatacgccagcggacgcggagcgagccggattgcttgtaaggacttcagcaccgtgcacgatcaccgggaccatgggacactttattctcgcccgatgcccgaaaggagagacgctgacagtgggatttacggacagcagaaagatcagccacacatgcacacacccgttccatcatgaaccacctgtgataggtagggagaggttccactctcgaccacaacatggtaaagagttaccttgcagcacgtacgtgcagagcaccgctgccactgctgaggagatagaggtgcatatgcccccagatactcctgaccgcacgctgatgacgcagcagtctggcaacgtgaagatcacagttaatgggcagacggtgcggtacaagtgcaactgcggtggctcaaacgagggactgacaaccacagacaaagtgatcaataactgcaaaattgatcagtgccatgctgcagtcactaatcacaagaattggcaatacaactcccctttagtcccgcgcaacgctgaactcggggaccgtaaaggaaagatccacatcccattcccattggcaaacgtgacttgcagagtgccaaaagcaagaaaccctacagtaacttacggaaaaaaccaagtcaccatgctgctgtatcctgaccatccgacactcttgtcttaccgtaacatgggacaggaaccaaattaccacgaggagtgggtgacacacaagaaggaggttaccttgaccgtgcctactgagggtctggaggtcacttggggcaacaacgaaccatacaagtactggccgcagatgtctacgaacggtactgctcatggtcacccacatgagataatcttgtactattatgagctgtaccccactatgactgtagtcattgtgtcggtggcctcgttcgtgcttctgtcgatggtgggcacagcagtgggaatgtgtgtgtgcgcacggcgcagatgcattacaccatatgaattaacaccaggagccactgttcccttcctgctcagcctgctatgctgcgtcagaacgaccaaggcggccacatattacgaggctgcggcatatctatggaacgaacagcagcccctgttctggttgcaggctcttatcccgctggccgccttgatcgtcctgtgcaactgtctgaaactcttgccatgctgctgtaagaccctggcttttttagccgtaatgagcatcggtgcccacactgtgagcgcgtacgaacacgtaacagtgatcccgaacacggtgggagtaccgtataagactcttgtcaacagaccgggttacagccccatggtgttggagatggagctacaatcagtcaccttggaaccaacactgtcacttgactacatcacgtgcgagtacaaaactgtcatcccctccccgtacgtgaagtgctgtggtacagcagagtgcaaggacaagagcctaccagactacagctgcaaggtctttactggagtctacccatttatgtggggcggcgcctactgcttttgcgacgccgaaaatacgcaattgagcgaggcacatgtagagaaatctgaatcttgcaaaacagagtttgcatcggcctacagagcccacaccgcatcggcgtcggcgaagctccgcgtcctttaccaaggaaacaacattaccgtagctgcctacgctaacggtgaccatgccgtcacagtaaaggacgccaagtttgtcgtgggcccaatgtcctccgcctggacaccttttgacaacaaaatcgtggtgtacaaaggcgacgtctacaacatggactacccaccttttggcgcaggaagaccaggacaatttggtgacattcaaagtcgtacaccggaaagtaaagacgtttatgccaacactcagttggtactacagaggccagcagcaggcacggtacatgtaccatactctcaggcaccatctggcttcaagtattggctgaaggaacgaggagcatcgctacagcacacggcaccgttcggttgccagattgcgacaaacccggtaagagctgtaaattgcgctgtggggaacataccaatttccatcgacataccggatgcggcctttactagggttgtcgatgcaccctctgtaacggacatgtcatgcgaagtaccagcctgcactcactcctccgactttgggggcgtcgccatcatcaaatacacagctagcaagaaaggtaaatgtgcagtacattcgatgaccaacgccgttaccattcgagaagccgacgtagaagtagaggggaactcccagctgcaaatatccttctcaacagccctggcaagcgccgagtttcgcgtgcaagtgtgctccacacaagtacactgcgcagccgcatgccaccctccaaaggaccacatagtcaattacccagcatcacacaccacccttggggtccaggatatatccacaacggcaatgtcttgggtgcagaagattacgggaggagtaggattaattgttgctgttgctgccttaattttaattgtggtgctatgcgtgtcgtttagcaggcactaa.

Regarding Chikungunya virus, another exemplary nucleotide sequence thatencodes a Capsid-envelope is described below (SEQ ID No.:7):

atggagttcatcccaacccaaactttttacaataggaggtaccagcctcgaccctggactccgcgccctactatccaagtcatcaggcccagaccgcgccctcagaggcaagctgggcaacttgcccagctgatctcagcagttaataaactgacaatgcgcgcggtaccacaacagaagccacgcaggaatcggaagaataagaagcaaaagcaaaaacaacaggcgccacaaaacaacacaaatcaaaagaagcagccacctaaaaagaaaccggctcaaaagaaaaagaagccgggccgcagagagaggatgtgcatgaaaatcgaaaatgattgtattttcgaagtcaagcacgaaggtaaggtaacaggttacgcgtgcctggtgggggacaaagtaatgaaaccagcacacgtaaaggggaccatcgataacgcggacctggccaaactggcctttaagcggtcatctaagtatgaccttgaatgcgcgcagatacccgtgcacatgaagtccgacgcttcgaagttcacccatgagaaaccggaggggtactacaactggcaccacggagcagtacagtactcaggaggccggttcaccatccctacaggtgctggcaaaccaggggacagcggcagaccgatcttcgacaacaagggacgcgtggtggccatagtcttaggaggagctaatgaaggagcccgtacagccctctcggtggtgacctggaataaagacattgtcactaaaatcacccccgagggggccgaagagtggagtcttgccatcccagttatgtgcctgttggcaaacaccacgttcccctgctcccagcccccttgcacgccctgctgctacgaaaaggaaccggaggaaaccctacgcatgcttgaggacaacgtcatgagacctgggtactatcagctgctacaagcatccttaacatgttctccccaccgccagcgacgcagcaccaaggacaacttcaatgtctataaagccacaagaccatacttagctcactgtcccgactgtggagaagggcactcgtgccatagtcccgtagcactagaacgcatcagaaatgaagcgacagacgggacgctgaaaatccaggtctccttgcaaatcggaataaagacggatgacagccacgattggaccaagctgcgttatatggacaaccacatgccagcagacgcagagagggcggggctatttgtaagaacatcagcaccgtgtacgattactggaacaatgggacacttcatcctggcccgatgtccaaaaggggaaactctgacggtgggattcactgacagtaggaagattagtcactcatgtacgcacccatttcaccacgaccctcctgtgataggtcgggaaaaattccattcccgaccgcagcacggtaaagagctaccttgcagcacgtacgtgcagagcaccgccgcaactaccgaggagatagaggtacacatgcccccagacacccctgatcgcacattaatgtcacaacagtccggcaacgtaaagatcacagtcaatggccagacggtgcggtacaagtgtaattgcggtggctcaaatgaaggactaacaactacagacaaagtgattaataactgcaaggttgatcaatgtcatgccgcggtcaccaatcacaaaaagtggcagtataactcccctctggtcccgcgtaatgctgaacttggggaccgaaaaggaaaaattcacatcccgtttccgctggcaaatgtaacatgcagggtgcctaaagcaaggaaccccaccgtgacgtacgggaaaaaccaagtcatcatgctactgtatcctgaccacccaacactcctgtcctaccggaatatgggagaagaaccaaactatcaagaagagtgggtgatgcataagaaggaagtcgtgctaaccgtgccgactgaagggctcgaggtcacgtggggcaacaacgagccgtataagtattggccgcagttatctacaaacggtacagcccatggccacccgcatgagataattctgtattattatgagctgtaccccactatgactgtagtagttgtgtcagtggccacgttcatactcctgtcgatggtgggtatggcagcggggatgtgcatgtgtgcacgacgcagatgcatcacaccgtatgaactgacaccaggagctaccgtccctttcctgcttagcctaatatgctgcatcagaacagctaaagcggccacataccaagaggctgcgatatacctgtggaacgagcagcaacctttgttttggctacaagcccttattccgctggcagccctgattgttctatgcaactgtctgagactcttaccatgctgctgtaaaacgttggcttttttagccgtaatgagcgtcggtgcccacactgtgagcgcgtacgaacacgtaacagtgatcccgaacacggtgggagtaccgtataagactctagtcaatagacctggctacagccccatggtattggagatggaactactgtcagtcactttggagccaacactatcgcttgattacatcacgtgcgagtacaaaaccgtcatcccgtctccgtacgtgaagtgctgcggtacagcagagtgcaaggacaaaaacctacctgactacagctgtaaggtcttcaccggcgtctacccatttatgtggggcggcgcctactgcttctgcgacgctgaaaacacgcagttgagcgaagcacacgtggagaagtccgaatcatgcaaaacagaatttgcatcagcatacagggctcataccgcatctgcatcagctaagctccgcgtcctttaccaaggaaataacatcactgtaactgcctatgcaaacggcgaccatgccgtcacagttaaggacgccaaattcattgtggggccaatgtcttcagcctggacacctttcgacaacaaaattgtggtgtacaaaggtgacgtctataacatggactacccgccctttggcgcaggaagaccaggacaatttggcgatatccaaagtcgcacacctgagagtaaagacgtctatgctaatacacaactggtactgcagagaccggctgtgggtacggtacacgtgccatactctcaggcaccatctggctttaagtattggctaaaagaacgcggggcgtcgctgcagcacacagcaccatttggctgccaaatagcaacaaacccggtaagagcggtgaactgcgccgtagggaacatgcccatctccatcgacataccggaagcggccttcactagggtcgtcgacgcgccctctttaacggacatgtcgtgcgaggtaccagcctgcacccattcctcagactttgggggcgtcgccattattaaatatgcagccagcaagaaaggcaagtgtgcggtgcattcgatgactaacgccgtcactattcgggaagctgagatagaagttgaagggaattctcagctgcaaatctctttctcgacggccttagccagcgccgaattccgcgtacaagtctgttctacacaagtacactgtgcagccgagtgccaccccccgaaggaccacatagtcaactacccggcgtcacataccaccctcggggtccaggacatctccgctacggcgatgtcatgggtgcagaagatcacgggaggtgtgggactggttgttgctgttgccgcactgattctaatcgtggtgctatgcgtgtcgttcagcaggcactaa.

In one embodiment, the present invention provides a vector comprisingthe nucleic acid molecule as described above, wherein the vectoroptionally comprises an expression control sequence operably linked tothe nucleic acid molecule.

Examples of an expression control sequence include, but are not limitedto, promoter such as CMV promoter, phage lambda PL promoter, the E. colilac, phoA and tac promoters, the SV40 early and late promoters, andpromoters of retroviral LTRs.

In this embodiment, the vector comprising an expression control sequenceoperably linked to the nucleic acid molecule as described above can beused as an expression vector for preparing the particle provided by thefirst aspect of the present invention.

The expression vectors can be prepared by a person skilled in the artbased on WO/2012/006180, the entire contents of which are incorporatedby reference herein.

Examples of vectors which can be used for expressing a virus likeparticle comprising a fusion protein of a polypeptide derived fromChikungunya virus (CHIKV) and a polypeptide of antigen include a vectorshown in VLP_CHI 512 vector (SEQ ID No.:8) containing CHIKV VLPpolynucleotide (SEQ ID No. 13; corresponding amino acid sequencerepresented by SEQ ID No.:14); and VLP_CHI 532 vector (SEQ ID No.: 9)containing CHIKV VLP polynucleotide (SEQ ID No. 15; corresponding aminoacid sequence represented by SEQ ID No.:16).

The expression vectors can be prepared by a person skilled in the artbased on US2012/0003266, the entire contents of which are incorporatedby reference herein.

Examples of vectors which can be used for expressing a virus likeparticle comprising a fusion protein of a polypeptide derived fromVenezuelan equine encephalitis virus (VEEV) and a polypeptide of antigeninclude a vector shown in VLP_VEEV VLP 518 vector (SEQ ID No.:10)containing VEEV VLP polynucleotide (SEQ ID No. 17; corresponding aminoacid sequence represented by SEQ ID No.:18); VLP_VEEV VLP 519 vector(SEQ ID No.11) containing VEEV VLP polynucleotide (SEQ ID No. 19;corresponding amino acid sequence represented by SEQ ID No.:20); andVLP_VEEV VLP 538 vector (SEQ ID No.: 12) containing VEEV VLPpolynucleotide (SEQ ID No. 21; corresponding amino acid sequencerepresented by SEQ ID No.:22).

In one embodiment, the present invention provides a nucleic acidmolecule which is designed for expression of a virus like particlecomprising a fusion protein of a polypeptide derived from Chikungunyavirus (CHIKV) or Venezuela equine encephalitis virus (VEEV) and apolypeptide derived from malaria antigen, which consists of a nucleotidesequence represented by SEQ ID Nos.26-27, 29-30, 32-33, 35-36, 38, 40 or42.

In one embodiment, the present invention provides a nucleic acidmolecule which is modified from the nucleic acid molecule having anucleotide sequence represented by any one of SEQ ID Nos.26-27, 29-30,32-33 or 35-36, 38, 40 or 42. The modified nucleic acid molecule mayhave at least 70%, 75%, 80%, 85%, 90%, 95% or 98% nucleotide sequenceidentity to the nucleic acid molecule having a nucleotide sequencerepresented by any one of SEQ ID Nos.26-27, 29-30, 32-33, 35-36, 38, 40or 42. Also, the modified nucleic acid molecule may be a mutant where atmost 10% of the amino acids are deleted, substituted, and/or added basedon the nucleic acid molecule having a nucleotide sequence represented byany one of SEQ ID Nos.26-27, 29-30, 32-33, 35-36, 38, 40 or 42.

(3) Composition or Vaccine

In the third aspect, the present invention provides a composition orvaccine comprising the particle provided in the first aspect of thepresent invention and/or the nucleic acid molecule provided in thesecond aspect of the present invention.

In one embodiment, the present invention provides a composition orvaccine comprising the Alphavirus or Flavivirus virus like particle(e.g. Chikungunya virus like particle or Venezuelan equine encephalitisvirus like particle) as described above or the nucleic acid molecule asdescribed above. The content of the Alphavirus or Flavivirus virus likeparticle and the content of the nucleic acid molecule may be 0.00001-1w/w %.

Dosage amount of the particle provided in the first aspect of thepresent invention (e.g. CHIKV VLP or VEEV VLP) may be 1-500 μg/day.

One or more malaria antigens may be used for one composition or onevaccine provided by the third aspect of the present invention.

The composition or vaccine may further comprise a pharmaceuticalacceptable carrier and/or adjuvant. Examples of adjuvant include, butare not limited to, aluminium salts, sodium hydroxide, Freund's completeadjuvant, Freund's incomplete adjuvant and Ribi solution (Sigma Adjuvantsystem, Sigma-Aldrich). The composition or vaccine provided in the thirdaspect of the present invention may contain buffering agent such asdibasic sodium phosphate hydrate, sodium dihydrogen phosphate and sodiumchloride; and preserving agent such as thimerosal. In one embodiment,the composition or vaccine is an aqueous solution containing 0.001-1 w/w% of the particle provided in the first aspect of the present invention(e.g. CHIKV VLP or VEEV VLP), 1-10 w/w % of buffering agent, 0.01-1 w/w% of adjuvant and 0.00001-0.001 w/w % of preserving agent.

A skilled person can prepare the pharmaceutical composition and vaccineusing conventional technique. For example, the particle provided in thefirst aspect of the present invention is mixed with buffer solutionhaving physiological pH (e.g. pH 5-9, pH7) to prepare the pharmaceuticalcomposition and vaccine provided in the third aspect of the presentinvention.

The pharmaceutical composition of the present invention may contain asingle active ingredient or a combination of two or more activeingredients, as far as they are not contrary to the objects of thepresent invention. For example, cytokines including chemokines,anti-body of cytokines such as anti TNF antibody (e.g. infliximab,adalimumab), anti-VEGF antibody (e.g. bevacizumab and ranibizumab),cytokine receptor antagonist such as anti HER2 antibody (e.g.Trastuzumab), anti EGF receptor antibody (e.g. Cetuximab), anti VEGFaptamer (e.g. Pegaptanib) and immunomodulator such as cyclosporine,tacrolimus, ubenimex may be used for the combination therapy.

In a combination of plural active ingredients, their respective contentsmay be suitably increased or decreased in consideration of theirtherapeutic effects and safety.

The term “combination” used herein means two or more active ingredientare administered to a patient simultaneously in the form of a singleentity or dosage, or are both administered to a patient as separateentities either simultaneously or sequentially with no specific timelimits, wherein such administration provides therapeutically effectivelevels of the two components in the body, preferably at the same time.

In one embodiment, the composition is a vaccine composition including aDNA vaccine. In one embodiment, the DNA vaccine provided by the presentinvention comprises CpG containing oligonucleotide.

The composition or vaccine provided in the third aspect of the presentinvention can be administered one or more times. When the composition orvaccine provided in the third aspect of the present invention isadministered more than one time, different particle provided in thefirst aspect of the present invention (e.g. CHIKV VLP or VEEV VLP) maybe used for each of the administration. In one embodiment, combinationof immunization using CHIKV VLP provided in the first aspect of theinvention and immunization using VEEV VLP provided in the first aspectof the invention is employed. For example, CHIKV VLP provided in thefirst aspect of the present invention may be used for the 1stimmunization and VEEV VLP provided in the first aspect of the presentinvention may be used for the 2nd immunization, or VEEV VLP provided inthe first aspect of the present invention may be used for the 1stimmunization and CHIKV VLP provided in the first aspect of the presentinvention may be used for the 2nd immunization.

A skilled person can determine timing of immunization using thecomposition or vaccine provided in the third aspect of the presentinvention. For example, 2nd immunization is performed 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 weeks after 1st immunization.

In one embodiment, the present invention provides a kit comprising

(a) a vaccine composition comprising the particle provided in the firstaspect of the present invention; and

(b) another vaccine composition comprising the particle provided in thefirst aspect of the present invention, wherein the particle contained in(a) is a virus like particle which is different from the particlecontained in (b). In this embodiment, the particle contained in (a) maybe Chikungunya virus like particle and the particle contained in (b) maybe Venezuelan equine encephalitis virus like particle.

In one embodiment, the present invention provides a kit comprising

(a) a vaccine composition comprising the particle provided in the firstaspect of the present invention; and

(b) another vaccine composition comprising the particle provided in thefirst aspect of the present invention,

(c) one or more vaccine composition, each of which comprises theparticle provided in the first aspect of the present invention,

wherein (a) is used for priming immunization and (b) and (c) are usedfor boosting immunization; and the particle contained in (a) is a viruslike particle which is different from the particle contained in (b); andthe particle contained in (c) is different from the particle containedin (a) and (b), or the same as the particle contained in (a) or (b).

The respective vaccine compositions contained in the above-described kitmay be administered simultaneously, separately or sequentially.

The Alphavirus or Flavivirus virus like particle (e.g. Chikungunya virusor Venezuelan equine encephalitis virus) provided in the first aspect ofthe present invention or the nucleic acid molecule provided by thesecond aspect of the invention can be used for the composition andvaccine provided in the third aspect of the present invention.

For example, Chikungunya or Venezuelan equine encephalitis virus likeparticle comprising or consisting of:

one or more (e.g. 240) capsid of Chikungunya virus (CHIKV) or Venezuelanequine encephalitis virus (VEEV);

one or more (e.g. 240) E1 of Chikungunya virus (CHIKV) or Venezuelanequine encephalitis virus (VEEV); and

one or more (e.g. 240) E2 of Chikungunya virus (CHIKV) or Venezuelanequine encephalitis virus (VEEV), wherein malaria antigen is insertedinto E2 of Chikungunya virus (CHIKV) or Venezuelan equine encephalitisvirus (VEEV) may be used for preparing the composition or vaccineprovided in the third aspect of the present invention. The E2 into whichthe antigen is inserted may consist of an amino acid sequencerepresented by SEQ ID No.50; the E1 may consist of an amino acidsequence represented by SEQ ID No.51; and the capsid may consist of anamino acid sequence represented by SEQ ID NO.: 52; orthe E2 into which the antigen is inserted may consist of an amino acidsequence represented by SEQ ID NO.53; the E1 may consist of an aminoacid sequence represented by SEQ ID NO.54; and the capsid may consist ofan amino acid sequence represented by SEQ ID NO.: 55.

The composition or vaccine provided in the third aspect of the presentinvention can be administered to a mammal (e.g. human) intramuscularly(i.m.), intracutaneously (i.c.), subcutaneously (s.c.), intradermally(i.d.) or intraperitoneally (i.p.).

The composition or vaccine provided in the third aspect of the presentinvention may be used for treating or preventing malaria.

Thus, use of the Alphavirus or Flavivirus (e.g. Chikungunya virus orVenezuelan equine encephalitis virus) virus like particle provided inthe first aspect of the present invention or the nucleic acid moleculeprovided by the second aspect of the invention for manufacturing apharmaceutical composition or vaccine for treating or preventing malariais also provided by the present invention.

(4) Method of Producing an Antibody, Method of Immunomodulation, Methodof Treating Malaria, Method of Inducing and/or Enhancing Immune ResponseAgainst a Malaria Antigen in a Mammal, Method of Passive Immunization,Method of Presenting an Antigen on Macrophage, and Method for Producinga Particle

In the fourth aspect, the present invention provides a method ofproducing an antibody, comprising contacting the particle provided inthe first aspect of the present invention and/or the nucleic acidmolecule provided in the second aspect of the present invention to amammal.

The antibody produced in the fourth aspect of the present invention maybe humanized using a conventional technique. Thus, in one embodiment,the method provided in the fourth aspect of the invention furthercomprises a step of humanizing non-human mammal produced antibody.

The particle provided in the first aspect of the present inventionand/or the nucleic acid molecule provided in the second aspect of thepresent invention may be administered directly into the patient, intothe affected organ or systemically, or applied ex vivo to cells derivedfrom the patient or a human cell line which are subsequentlyadministered to the patient, or used in vitro to select a subpopulationfrom immune cells such as B-cell and T-cell derived from the patient,which are then re-administered to the patient.

According to the present invention, the virus like particle can beapplied for the immune therapy.

In the fifth aspect, the present invention provides a method ofimmunomodulation, a method of treating malaria, a method of inducingand/or enhancing immune response against a malaria antigen in a mammalcomprising administering the composition provided in the third aspect ofthe present invention to a mammal.

In sixth aspect, the present invention provides a method of passiveimmunization against a malaria-causing pathogen, comprisingadministering the antibody provided in the fourth aspect of the presentinvention to a mammal.

In seventh aspect, the present invention provides a method of presentinga malaria antigen on macrophage, comprising contacting the particleprovided in the first aspect of the present invention and/or the nucleicacid molecule provided in the second aspect of the present invention toa mammal.

In eighth aspect, the present invention provides a method for producingthe particle provided in the first aspect of the present invention,comprising preparing a vector designed for expression of said particle;culturing a cell which is transfected with said vector to express saidparticle; and recovering said particle.

Examples of mammal include, but are not limited to, a human.

In one embodiment, the present invention provides a method of producingan antibody against malaria antigen, comprising contacting theChikungunya or Venezuelan equine encephalitis virus like particle asdescribed above and/or the nucleic acid molecule as described above to amammal. The produced antibody may be an antibody which can specificallybind to a malaria antigen comprised in the Chikungunya or Venezuelanequine encephalitis virus like particle or a malaria antigen encoded bythe nucleic acid molecule. The method of producing an antibody providedby the present invention may be a useful for producing a monoclonal orpolyclonal antibody against a malaria antigen.

In one embodiment, an antibody against malaria antigen obtained by themethod of producing an antibody according to the present invention isused for passive immunization. The method of passive immunization maycomprise administering the obtained antibody to a mammal.

In one preferred embodiment, the immunomodulation provided by thepresent invention is inducing and/or enhancing immune response againstmalaria antigen in a mammal. Thus, in one embodiment, the presentinvention provides a method of inducing and/or enhancing immune responseagainst malaria antigen in a mammal, comprising administering aneffective amount of the composition as described above to the mammal.

Given the symptom of patients infected with Chikungunya or Venezuelanequine encephalitis together with unusual big molecule of Chikungunya orVenezuelan equine encephalitis, this VLP can act effectively andefficiently to target macrophage and its composition such as cytokinesand immunomodulative compounds.

In one aspect, the present invention provides a method of presenting anantigen on macrophage, comprising administering the Chikungunya orVenezuelan equine encephalitis virus like particle as described aboveand/or the nucleic acid molecule as described above to a mammal. TheChikungunya or Venezuelan equine encephalitis virus like particleprovided by the present invention is good to target macrophage. In oneembodiment, the Chikungunya or Venezuelan equine encephalitis virus likeparticle provided by the present invention is a kind of delivery systemof the at least one antigen, which is comprised in the Chikungunya orVenezuelan equine encephalitis virus like particle, to macrophage.

In one embodiment, the present invention provides a method for producingChikungunya or Venezuelan equine encephalitis virus like particleprovided in the first aspect of the present invention, comprisingpreparing a vector designed for expression of said particle; culturing acell which is transfected with said vector to express said particle; andrecovering said particle. In this embodiment, transfection can beconducted using a conventional method. Cells using for the transfectionmay be 293 cells. Recovering VLP may include collecting a conditionedmedium after cells are transfected with a vector, and may furtherinclude purify VLP from the conditioned medium usingultracentrifugation.

The present invention will be described in detail with reference to thefollowing example, which, however, is not intended to limit the scope ofthe present invention.

EXAMPLES Example 1 Preparation of Chikungunya Virus (CHIKV) LikeParticle Comprising a Virus Structural Polypeptide and a Fragment ofMalaria Antigen

The following polynucleotides of malaria CSP1 proteins are used. Nterminal linker is SGG and C terminal linker is GGS.

VLP74 (6 repeat of NPNA amino acid sequence) (SEQ ID No.: 46)Sggnpnanpnanpnanpnanpnanpnaggs (SEQ ID No.: 47)(Tccggaggaaacccgaatgccaatcccaacgcgaaccccaatgctaacccaaatgccaacccaaacgccaaccccaacgctggtggatcc)VLP78 (25 repeat of NPNA amino acid sequence) (SEQ ID No.: 48Sggnpnanpnanpnanpnanpnanpnvdpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpn anpnaggs(SEQ ID No.: 49) (tccggaggaaacccgaatgccaatcccaacgcgaaccccaacgctaaccccaacgccaatccgaatgcaaacccgaacgttgacccaaacgccaacccgaatgccaatcccaacgcgaaccccaatgctaacccaaatgccaacccaaacgccaaccccaacgctaatccaaacgccaaccctaacgccaatcccaacgcgaatcctaacgctaatcccaacgcaaatcccaatgctaatccgaacgcgaaccctaatgcaaaccccaacgccaacccgaacgctaacccgaacgctaatcccaacgccggtggatcc)

The respective polynucleotides was inserted between the codons encodingSer at 531-position and Asn at 532-position of SEQ ID Nos.15 or 16 (SEQID Nos.1 or 2) to construct a plasmid (hereinafter referred to asCHIKV-VLP74 or 78) for expressing Chikungunya virus like particle wherethe modified VLP74 or 78-derived peptide is inserted into E2 ofChikungunya virus structural polypeptide.

293F cells (Lifetechnology) were transfected with the plasmid using PEI(GE Healthcare) or GeneX (ATCC). 4 days after the transfection, theconditioned medium was collected and centrifuged at 3000 rpm for 15minutes to separate it from cells. The supernatant was filtrated using0.45 μm filter to obtain virus like particles. The virus like particleswere concentrated using TFF column and purified using QXL column (GEHealthcare) to obtain purified virus like particles. When animals wereimmunized with virus like particles, the purified virus like particleswere further concentrated using spin column (Molecular Weight-cutoff:100 kDa) to prepare the virus like particles for the immunization.

The expression of VLP comprising VLP74 or 78 conjugated with Chikungunyavirus structural polypeptide was confirmed by Western Blot using anantibody specific for CHIKV (ATCC: VR-1241AF) and an antibody specificfor VLP74 or 78.

Example 2 Preparation of Venezuelan Equine Encephalitis Virus (VEEV)Like Particle Comprising a Virus Structural Polypeptide and a Fragmentof Malaria Antigen

The same polynucleotides of malaria CSP1 proteins (VLP74 and VLP78) usedin EXAMPLE 1 are used. N terminal linker is SGG and C terminal linker isGGS.

The respective polynucleotides was inserted between the codons encodingSer at 518-position and Ser at 519-position of SEQ ID Nos.19 or 20 (SEQID No.3) to construct a plasmid (hereinafter referred to as VEEV-VLP74or 78) for expressing Venezuelan equine encephalitis virus like particlewhere the modified VLP74 or 78-derived peptide is inserted into E2 ofVenezuelan equine encephalitis virus structural polypeptide.

293F cells (Lifetechnology) were transfected with the plasmid using PEI(GE Healthcare) or GeneX (ATCC). 4 days after the transfection, theconditioned medium was collected and centrifuged at 3000 rpm for 15minutes to separate it from cells. The supernatant was filtrated using0.45 μm filter to obtain virus like particles. The virus like particleswere concentrated using TFF column and purified using QXL column (GEHealthcare) to obtain purified virus like particles. When animals wereimmunized with virus like particles, the purified virus like particleswere further concentrated using spin column (Molecular Weight-cutoff:100 kDa) to prepare the virus like particles for the immunization.

The expression of VLP comprising VLP 74 or 78 conjugated with Venezuelanequine encephalitis virus structural polypeptide was confirmed byWestern Blot using an antibody specific for VEEV and an antibodyspecific for VLP74 or 78.

Example 3 Immunogenicity in Non-Human Primate (Monkey)

The monkeys were immunized with ×25-CHI (80 ug) at 0 week and ×6-VEE (80ug) at 3 week by intramuscular injection with or without adjuvant (SigmaAdjuvant System, Sigma, S6322). ×25-CHI means 25 times malaria CSPrepeat amino acid NPNA on CHIKV VLP particle (VLP78_15). ×6-VEE means 6times malaria CSP repeat amino acid NPNA on VEEV VLP particle(VLP74_25). The blood is taken at 2 and 5 weeks after the firstimmunization.

96 well ELISA plate were coated with 50 ng of RecombinantCircumsporozoite Protein (rCSP) (Reagent Proteins, ATG-422) in 100 ulPBS buffer pre well. The Plates after 2 hours incubation were washedthree times TBS buffer containing 0.05% Tween-20 and blocked with TBSbuffer containing 0.05% Tween-20 and 5% dry milk. The heat inactivateddiluted serum from monkeys were added in the blocking buffer andincubated for 1 h at room temperature. After washing three times,peroxidase labeled goat anti-human IgG or anti-mouse IgG was added at1:4000 dilution and incubated for 1 h at room temperature. After washingthree times, Peroxidase substrate was added for development andincubated for 10 mins and 2N H2SO4 was added to stop the development.The data were analyzed using Gen5 (BioTek) and GraphPad Prism6 (GraphPadsoftware Inc).

The Immunogenicities are shown in FIGS. 4 to 6.

Induction of antibodies against CSP was found in the serum of allmonkeys immunized with Malaria VLPs (see FIG. 4). The mean OD valuesindicating titer of antibodies against CSP is shown in FIG. 5. FIG. 5shows that the serum from immunized monkeys induced high titer ofantibodies against CSP.

As seen in FIG. 6, higher titer of antibodies against CSP was achievedwhen CHIKV VLP particle comprising NPNA and VEEV VLP particle comprisingNPNA were used for the priming immunization and boosting immunization,respectively, compared to when only CHIKV VLP particle comprising NPNAwas used for both of the priming immunization and the boostingimmunization. In addition, FIG. 6 shows that use of adjuvant furtherenhanced the titer of antibodies against CSP. Further, FIG. 6 shows thatadministration of 25-repeats of NPNA induces higher titer of antibodiesagainst CSP compared to administration of 6-repeats of NPNA.

The anti-Pf CSP antibody titer in the serum from the monkeys immunizedwith ×25-CHI (80 ug) at 0 week and ×6-VEE (80 ug) at 3 week withoutusing adjuvant was measured by ELISA at Malaria Serology Lab MalariaVaccine Branch, WRAIR. In the ELISA performed at Malaria Serology LabMalaria Vaccine Branch, WRAIR, the plates were coated with CSPrp((NPNA)6 Peptide) [0.2 μg/μL] (Supplier: Eurogentec EP070034) and Goatα-Human IgG (KPL/074-1002 LOT#120714) was used as 2nd antibody. Thefinal titer was determined by the dilution factor that yields an OD of1.0 (414 nm).

As a result, the anti-Pf CSP antibody titer in the serum from themonkeys was enhanced after 2nd immunization compared to 1st immunization(see Table 2). Compared to the anti-Pf CSP antibody titer in the serumfrom the monkeys immunized with RTS,S (GlaxoSmithKline), the anti-Pf CSPantibody titer in the serum from the monkeys immunized with ×25-CHI (80ug) and ×6-VEE (80 ug) in the absence of adjuvant was considered to behigher even though RTS,S (GlaxoSmithKline) contains adjuvant.

TABLE 2 After 1st immunization After 2nd immunization Animal No. Week 2Week 5 1 8990 29420 2 48210 44100 3 80400 51230 4 16260 19640 Geometricmean 27359 33801

Example 4 Immunogenicity in Mouse

The mice immunized with 10 ug of VLP78_15 at week 0, 10 ug of VLP74_25at week 3 and 10 ug of VLP78_15 at week 6 with or without adjuvant(Sigma Adjuvant System, Sigma, S6322) by intramuscular injection.

The anti-Pf CSP antibody titer in the serum from the immunized mice weremeasured by ELISA at Malaria Serology Lab Malaria Vaccine Branch, WRAIR,where plates were coated with CSPrp ((NPNA)6 Peptide) [0.2 μg/μL](Supplier: Eurogentec EP070034) and Goat α-Mouse IgG (KPL/074-1806LOT#100737) is used as 2nd antibody to detect the antibodies in theserum.

The final titer was determined by the dilution factor that yields an ODof 1.0 (414 nm).

The Immunogenicity are shown in Tables 3 and 4.

Tables 3 and 4 show that higher titer of antibodies against CSP wasachieved after immunizing virus like particle three times. In addition,Tables 3 and 4 show that use of adjuvant enhanced the titer ofantibodies against CSP.

TABLE 3 Mouse Week 3 (after 1^(st) immunization) Mouse No. VLP VLP +Adjuvant 1 6070 QNS 2 5850 13680 3 9610 7610 4 5440 23370 5 16320 27390Geometric Mean 7875 16066 QNS = Quantity not sufficient to test

TABLE 4 Mouse Week 9 (after 1st immunization) Mouse No. VLP VLP +Adjuvant 1 35510 729000 2 15040 197800 3 41650 106700 4 37250 436000 548200 497600 Geometric Mean 33134 319666

Example 5 Immunogenicity of P. yoelii CSP Inserted VLP in Mice

QGPGAP seen in the rodent malaria CSP (P. yoelii CSP) was used as anantigen. 6×QGPGAP was inserted into CHIKV VLP. The mice were immunizedwith the CHIKV VLP 2 times at 0 and 8 week (20 ug VLP per mouse) byintramuscle injection with or without Adjuvant Ribi.

Immunogenicity was confirmed at 4, 6, 10 and 14 weeks after the firstimmunization. The anti-P. yoelii CSP antibody were measured by ELISA.The ELISA was performed in the same way as ELISA described in Example 3except that the plates were coated with P. Yoelii CSP repeat sequencepeptide at 0.1 ng/μl. The secondary antibody was anti-mouse IgG HRP(Cell signal, #7076S). The results are shown in FIGS. 7-9.

FIGS. 7-9 show that higher titer of antibodies against CSP was achievedby intramuscler administration of CHIKV VLP comprising 6×QGPGAP. Inaddition, FIGS. 7-9 show that use of adjuvant enhanced the titer ofantibodies against CSP.

Example 6 Protection of Mice Against Malaria by Intramuscle Injection ofCHIKV VLP Comprising P. yoelii CSP Epitope: 6×QGPGAP

6×QGPGAP was inserted into CHIKV VLP. The mice (n=5) were immunized withthe CHIKV VLP 2 times at 0 and 8 week (20 ug VLP per mouse) byintramuscle injection with or without Adjuvant Ribi (see FIG. 10).Rodent malaria: P. yoelii-challenge (i.v.) was conducted at 17 weeks(see FIG. 10).

Malaria infection was confirmed by PCR. Genomic DNA was purified fromthe mice blood day 6 after challenge. 18S malaria DNA was amplified byPCR. FIG. 11 shows results of the PCR, indicating that all of 5 controlmice (PBS injection) were infected with malaria; all of 5 mice immunizedwith Control VLP were infected with malaria; among 5 mice immunized withCHIKV VLP comprising 6×QGPGAP, 2 mice were infected with malaria and 3mice were not infected with malaria; and among 5 mice immunized withCHIKV VLP comprising 6×QGPGAP with adjuvant: Ribi, 1 mouse was infectedwith malaria and 4 mice were not infected with malaria.

Example 7 Preparation of Vaccine Composition Comprising ChikungunyaVirus (CHIKV) Like Particle Comprising NPNA Repeat or Venezuelan EquineEncephalitis Virus (VEEV) Like Particle Comprising NPNA Repeat

Chikungunya virus (CHIKV) like particle comprising 6× or 25×NPNA wasprepared according to Example 1, and Venezuelan equine encephalitisvirus (VEEV) like particle comprising 6×NPNA was prepared according toExample 2. To prepare a vaccine composition, 80 μg of each of theprepared particles was mixed with 1 ml of Sucrose Phosphate Solution, pH7.2, Endotoxin Free (Teknova, SP buffer).

The invention claimed is:
 1. A Chikungunya virus (CHIKV) or Venezuelanequine encephalitis virus (VEEV) virus-like particle, wherein saidvirus-like particle contains at least one malaria antigen inserted intoan E2 envelope protein to form a fusion protein, wherein said at leastone malaria antigen is inserted within a CHIKV E2 envelope protein at asite that corresponds in position to a site between residues 509-512,the site between residues 519-520, the site between residues 529-530, orthe site between residues 531-532 of SEQ ID NO: 1 or 2; or wherein saidat least one antigen is inserted within a VEEV E2 envelope protein at asite that corresponds in position to a site between residues 515-520, ora site between residues 536-539 of SEQ ID NO: 3, and wherein said atleast one malaria antigen is selected from the group consisting of (a)and (b): (a) (NPNA)_(n), wherein n is from 4 to 30; and (b)(EYLNKIQNSLSTEWSPCSVT)_(y), wherein y is from 1 to
 6. 2. The virus-likeparticle according to claim 1, which comprises capsid, E1 and E2.
 3. Thevirus-like particle according to claim 1, wherein the virus-likeparticle comprises E1, E2 and capsid protein, wherein said envelopeprotein E2 consists of the amino acid sequence of SEQ ID NO: 50; whereinsaid envelope protein E1 consists of the amino acid sequence of SEQ IDNO: 51; and wherein said capsid protein consists of the amino acidsequence of SEQ ID NO:
 52. 4. The particle according to claim 1, whereinthe particle is Venezuelan equine virus like particle consisting of oneor more envelope protein E2 into which the antigen is inserted, one ormore envelope protein E1 and one or more capsid, and wherein theenvelope protein E2 into which the antigen is inserted consists of anamino acid sequence represented by SEQ ID NO.53; the envelope protein E1consists of an amino acid sequence represented by SEQ ID NO.54; and thecapsid consists of an amino acid sequence represented by SEQ ID NO.: 55.5. An isolated nucleic acid molecule comprising a nucleotide forexpressing the particle according to claim
 1. 6. An isolated nucleicacid molecule consisting of a nucleotide sequence which has a sequenceidentity of 90% or more with a nucleotide sequence represented by SEQ IDNos.26-27, 29-30, 32-33, 35-36, 38, 40 or
 42. 7. A vector comprising thenucleic acid molecule according to claim 5, wherein the vectoroptionally comprises an expression control sequence operably linked tothe nucleic acid molecule.
 8. A pharmaceutical composition comprising:particle according to claim 1 and/or a nucleic acid molecule comprisinga nucleotide sequence for expressing the particle according to claim 1;and (b) a pharmaceutically acceptable carrier.
 9. A vaccine compositioncomprising the virus-like according to claim
 1. 10. A method of treatingmalaria, comprising administering an effective amount of the compositionof claim 8 to a mammal.
 11. A vaccine comprising, as separatecomponents, a priming composition comprising at least one first malariaantigen or at least one polynucleotide encoding at least one firstmalaria antigen; and a boosting composition comprising at least onepolypeptide comprising at least one second malaria antigen or at leastone polynucleotide encoding at least one second malaria antigen, whereinat least one of said compositions comprises the particle of claim 1, ora nucleic acid encoding said particle.
 12. The vaccine of claim 11,wherein the polynucleotide encodes substantially all of thecircumsporozoite protein.
 13. The vaccine composition according to claim9, for use in the prevention or treatment of malaria.
 14. A kitcomprising (a) a first vaccine composition comprising a virus-likeparticle according to claim 1; and (b) a second vaccine compositioncomprising a virus-like particle according to claim 1, wherein thevirus-like particle contained in said first vaccine composition is avirus-like particle which is different from the virus-like particlecontained in said second vaccine composition.
 15. The kit according toclaim 14, further comprising (c) a third vaccine composition comprisinga virus-like particle according to claim 1, wherein said first vaccinecomposition is used for priming immunization and said second and thirdvaccine compositions are used for boosting immunization, and wherein thevirus-like particle contained in said third vaccine composition isdifferent from the virus-like particle contained in said first andsecond vaccine compositions, or is the same as a virus-like particlecontained in either said first or second vaccine composition.
 16. Theisolated nucleic acid molecule according to claim 6, which consists of anucleotide sequence represented by SEQ ID Nos. 26-27, 29-30, 32-33,35-36, 38, 40 or
 42. 17. The virus-like particle according to claim 1,wherein one or two linkers intervene between the N-terminal residue ofthe at least one malaria antigen and the E2 envelope protein, and/orbetween the C-terminal residue of the at least one malaria antigen andthe E2 envelope protein.
 18. The virus-like particle according to claim1, wherein the virus-like particle comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 28, 31, 39 and 41.